Early work in the field of chromatography utilized supports prepared from natural substances such as agarose, cross-linked dextran and the like. These materials, while adequate, lack rigidity, and therefore are not particularly useful for high pressure chromatography because the pressure thereon can deform the substances, or otherwise cause them to collapse, thereby slowing the acceptable flow rate considerably.
More recently, new developments have been made in the field allowing the functionalization of rigid support materials. Among the chromatographic supports used in the separation of biological macromolecules are bonded phase silica and porous glass beads comprising on their surface pendant hydrophilic moeities resulting from reaction of the activated silica or glass surface with a reactive organosilane, immobilizing the organosilane through a siloxane linkage to a silanol group on the surface. The hydrophilic moeity is attached to the reactive organosilane through an alkyl or aryl spacer group. Functional groups including glycidoxypropyl, aminopropyl, polyethyleneoxide and polyethyleneimine and derivatives thereof, have been used.
Certain problems were encountered in using these prior art systems, particularly for chromatographic separation of biological macromolecules. Nonspecific hydrophobic interaction between the straight chain alkyl backbone and the hydrophobic portion of the biological molecules has been observed, particularly when chromatography occurs under aqueous conditions. In addition, ionic interaction and hydrogen bonding has been observed between the silanol matrix and the polar functional groups of the derivatizing silane. This causes the functional groups to double back and bind to the matrix thereby making them unavailable for the intended chromatographic interaction.
In an effort to prepare materials lacking the aforementioned properties, bonded phase chromatographic supports prepared by reaction of 3-glycidoxypropyltrimethoxysilane with activated particulate silica or controlled pore glass were further derivatized by the addition of an amine or other nucleophile to the epoxide functionality of the bonded phase, as shown in Equation A below: ##STR1##
Other approaches have included the preparation of phases resulting from the adsorption of polyethyleneimine and polyethyeneoxide to particulate silica followed by derivatization of the polymeric chain.
Derivatives prepared from glycidoxypropyl bonded phases exhibit considerable hydrophobic secondary interactions. Polyether bonded phases exhibit similar problems. These materials require mobile phase additives to mask residual silanol activity and hydrophobic interaction.
Most recently, in an effort to prepare chromatographic support suitable for the separation of biological macromolecules, glycidoxypropyl and methacrylate bonded phases have been copolymerized with acrylamide or functionalized acrylamide.